This invention relates to the field of molecular biology and microbiology. More specifically, this invention describes a new, genetically engineered biocatalyst possessing enhanced tyrosine ammonia-lyase activity.
Phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) is widely distributed in plants (Koukol et al., J. Biol. Chem. 236:2692-2698 (1961)), fungi (Bandoni et al., Phytochemistry 7:205-207 (1968)), yeast (Ogata et al., Agric. Biol. Chem. 31:200-206 (1967)), and Streptomyces (Emes et al., Can. J. Biochem. 48:613-622 (1970)), but it has not been found in Escherichia coli or mammalian cells (Hanson and Havir In The Enzymes, 3rd ed.; Boyer, P., Ed.; Academic: New York, 1967; pp 75-167). PAL is the first enzyme of phenylpropanoid metabolism and catalyzes the removal of the (pro-3S)-hydrogen and xe2x80x94NH3+ from L-phenylalanine to form trans-cinnamic acid. In the presence of a P450 enzyme system, trans-cinnamic acid can be converted to para-hydroxycinnamic acid (PHCA) which serves as the common intermediate in plants for production of various secondary metabolites such as lignin and isoflavonoids. In microbes however, cinnamic acid and not the PHCA acts as the precursor for secondary metabolite formation. No cinnamate hydroxylase enzyme has so far been characterized from microbial sources. The PAL enzyme in plants is thought to be a regulatory enzyme in the biosynthesis of lignin, isoflavonoids and other phenylpropanoids (Hahlbrock et al., Annu. Rev. Plant Phys. Plant Mol. Biol. 40:347-369 (1989)). However, in the red yeast, Rhodotorula glutinis (Rhodosporidium toruloides), this lyase degrades phenylalanine as a catabolic function and the cinnamate formed by the action of this enzyme is converted to benzoate and other cellular materials.
The gene sequence of PAL from various sources, including Rhodosporidium toruloides, has been determined and published (Edwards et al., Proc. Natl. Acad. Sci., USA 82:6731-6735 (1985); Cramer et al., Plant Mol. Biol. 12:367-383 (1989); Lois et al., EMBO J. 8:1641-1648 (1989); Minami et al., Eur. J. Biochem. 185:19-25 (1989); Anson et al., Gene 58:189-199 (1987); Rasmussen and Oerum, DNA Sequence, 1:207-211 (1991). The PAL genes from various sources have been over-expressed as active PAL enzyme in yeast, Escherichia coli and insect cell culture (Faulkner et al., Gene 143:13-20 (1994); Langer et al., Biochemistry 36:10867-10871 (1997); McKegney et al., Phytochemistry 41:1259-1263 (1996)). PAL has received attention because of its potential usefulness in correcting the inborn error of metabolism phenylketonuria (Bourget et al., FEBS Lett. 180:5-8 (1985); U.S. Pat. No. 5,753,487), in altering tumor metabolism (Fritz et al. J. Biol. Chem. 251:4646-4650 (1976)), in quantitative analysis of serum phenylalanine (Koyama et al., Clin. Chim. Acta, 136:131-136 (1984)) and as a route for synthesizing L-phenylalanine from cinnamic acid (Yamada et al., Appl. Environ. Microbiol. 42:773 (1981), Hamilton et al., Trends in Biotechnol. 3:64-68 (1985) and Evans et al., Microbial Biotechnology 25:399-405 (1987)).
In plants, the PAL enzyme converts phenylalanine to trans-cinnamic acid which in turn is hydroxylated at the para position by cinnamate-4-hydroxylase to make PHCA (Pierrel et al., Eur. J. Biochem. 224:835 (1994); Urban et al., Eur. J Biochem. 222:843 (1994); Cabello-Hurtado et al., J. Biol. Chem. 273:7260 (1998); and Teutsch et al., Proc. Natl. Acad. Sci. USA 90:4102 (1993)). However, since further metabolism of cinnamic acid in microbial systems does not usually involve its para hydroxylation to PHCA, information regarding this reaction in microorganisms is scarce.
Information available indicates that PAL from some plants and micro-organisms, in addition to its ability to convert phenylalanine to cinnamate, can accept tyrosine as substrate. In such reactions the enzyme activity is designated tyrosine ammonia lyase (TAL). Conversion of tyrosine by TAL results in the direct formation of PHCA from tyrosine without the intermediacy of cinnamate. However, all natural PAL/TAL enzymes prefer to use phenylalanine rather than tyrosine as their substrate. The level of TAL activity is always lower than PAL activity, but the magnitude of this difference varies over a wide range. For example, the parsley enzyme has a KM for phenylalanine of 15-25 xcexcM and for tyrosine 2.0-8.0 mM with turnover numbers 22/sec and 0.3/sec respectively (Appert et al., Eur. J. Biochem. 225:491 (1994)). In contrast, the maize enzyme has a KM for phenylalanine only fifteen times higher than for tyrosine, and turnover numbers about ten-fold higher (Havir et al., Plant Physiol. 48:130 (1971)). The exception to this rule, is the yeast, Rhodosporidium, in which a ratio of TAL catalytic activity to PAL catalytic activity is approximately 0.58 (Hanson and Havir In The Biochemistry of Plants; Academic: New York, 1981; Vol. 7, pp 577-625).
The above mentioned biological systems provide a number of enzymes that may be useful in the production of PHCA, however, the efficient production of this monomer has not been achieved. The problem to be overcome therefore is the design and implementation of a method for the efficient production of PHCA from a biological source using an inexpensive substrate or fermentable carbon source. Applicants have solved the stated problem by engineering both microbial and plant hosts to produce PHCA, either by the overexpression of foreign genes encoding PAL and p450/p-450 reductase system or by the expression of genes encoding mutant and wildtype TAL activity.
The object of the present invention is bioproduction of PHCA, a compound that has potential as a monomer for production of Liquid Crystal Polymers (LCP). There are two potential bio-routes for production of PHCA from glucose and other fermentable carbon substrates:
1) Conversion of phenylalanine to cinnamic acid to PHCA. This route requires the enzyme PAL as well as a cytochrome P-450 and a cytochrome P-450 reductase (Scheme 1).
2) Conversion of tyrosine to PHCA in one step without the intermediacy of cinnamate (Scheme 1). This route requires the enzyme TAL which is likely to be very similar to PAL but with a higher substrate specificity for tyrosine. This route does not require the cytochrome P-450 and the cytochrome P-450 reductase. Operation of the TAL route therefore requires generation of a biocatalyst with increased TAL activity to function through the TAL route. 
The present invention describes methods for bioproduction of PHCA through conversion of: 1) cinnamate to PHCA; 2) glucose to phenylalanine to PHCA via the PAL route and 3) through generation of a new biocatalyst possessing enhanced tyrosine ammonia-lyase (TAL) activity. The evolution of TAL requires isolation of a yeast PAL gene, mutagenesis and evolution of the PAL coding sequence, and selection of variants with improved TAL activity. The instant invention further demonstrates the bioproduction of PHCA from glucose through the above mentioned routes in various fungi and bacteria.
It is an object of the present invention therefore to provide a method for the production of PHCA comprising: (i) contacting a recombinant host cell with a fermentable carbon substrate, said recombinant cell lacking a P-450/P-450 reductase system and comprising a gene encoding a tyrosine ammonia lyase activity operably linked to suitable regulatory sequences (ii) growing said recombinant cell for a time sufficient to produce PHCA; and (iii) optionally recovering said PHCA. Within the context of the invention a fermentable carbon substrate may be selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, carbon dioxide, methanol, formaldehyde, formate, and carbon-containing amines and the host cell from the group consisting of bacteria, yeasts, filamentous fungi, algae and plant cells.
Similarly provided are recombinant host cells lacking a cytochrome P-450/P-450 reductase system and comprising a gene encoding a tyrosine ammonia lyase activity operably linked to suitable regulatory sequences.
Additionally provided is a method for the production of PHCA comprising: (i) contacting a recombinant yeast cell with a fermentable carbon substrate, said recombinant cell comprising: a) a gene encoding a plant P-450/P-450 reductase system; and b) a gene encoding a yeast PAL activity operably linked to suitable regulatory sequences; (ii) growing said recombinant cell for a time sufficient to produce PHCA; and (iii) optionally recovering said PHCA.
It is another object of the present invention to provide a method for identifying a gene encoding a TAL activity comprising: (i) contacting a recombinant microorganism comprising a foreign gene suspected of encoding a TAL activity with PHCA for a time sufficient to metabolize PHCA; and (ii) monitoring the growth the recombinant microorganism whereby growth of the organism indicates the presence of a gene encoding a TAL activity.
Similarly a method for identifying a gene encoding a TAL activity is provided comprising: (i) transforming a host cell capable of using PHCA as a sole carbon source with a gene suspected of encoding a TAL activity to create a transformant; (ii) comparing the rate of growth of the transformant with an untransformed host cell capable of using PHCA as a sole carbon source wherein an accelerated rate of growth by the transformant indicates the presence of a gene encoding a TAL activity.
Additionally the present invention provides an isolated nucleic acid fragment selected from the group consisting of: a) an isolated nucleic acid fragment encoding a truncated mutant tyrosine ammonia lyase polypeptide, the polypeptide having the amino acid sequence as set forth in SEQ ID NO:32; b) an isolated nucleic acid fragment have the nucleotide sequence as set forth in SEQ ID NO:31; and c) an isolated nucleic acid fragment completely complementary to either (a) or (b), and polypeptides encoded by the same.
Similary the invention provides an isolated nucleic acid fragment selected from the group consisting of: a) an isolated nucleic acid fragment encoding a mutant tyrosine ammonia lyase polypeptide, the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ DI NO:36, SEQ ID NO:37 and SEQ ID NO:38; and b) an isolated nucleic acid fragment completely complementary to either (a), and polypeptides encoded by the same.